Multiple exciting light system

ABSTRACT

A multiple exciting light system for a test of a biological sample labeled with fluorochrome includes a case, in which a sample table, a first light source module, a second light source module, and filter unit are provided. The sample table is provided in the case to put the biological sample thereon. The first light source module is provided in the case to emit visible light as a first light; and the second light source module is provided in the case to emit visible light or invisible light as a second light. The first light from the first light source module and the second light from the second light source module excite the fluorochrome in the biological sample at the same time to generate a third light with a third wavelength by fluorescence resonance superposition energy transfer.

The current application claims a foreign priority to the patentapplication of Taiwan No. 100137934 filed on Oct. 19, 2011.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to a light system, and moreparticularly to a multiple exciting light system for observation ofbiological samples in the biological test.

2. Description of the Related Art

With advancement in biotechnology, we pay much attention to test thebiological sample, such as protein, cell, and deoxyribonucleic acid(DNA). In prior art, the biological sample is tested by fluorescencedetection. In fluorescence detection, fluorochrome has an excitationstate and an emission state to mark the specified molecules in thebiological sample.

Take the DNA molecule for example, it is added in an electrophoresissolution, which includes buffer solution, such as TAE buffer, and gel,such as agarose gel electrophoresis (AGE) or polyacrylamide gelelectrophoresis (PAGE). The electrophoresis solution is supplied withvoltage to form gel electrophoresis and obtain a DNA gel. Next, the DNAgel is stained with fluorochrome, such as ethidium bromide (EtBr). Now,the DNA gel may be exposed to UV light to excite the fluorochrome togenerate fluorescence so that researchers may observe the DNA in agarosegel or polyacryamide gels through fluorescence. However, UV light mustbe operated in a dark room. Besides, it is known that UV light isharmful to human skin. It is bad for the researchers who are exposed tothe UV light for a long time in the test.

In conclusion, the present invention will introduce a multiple excitinglight system to overcome the drawbacks as described above.

SUMMARY OF THE INVENTION

The primary objective of the present invention is to provide a multipleexciting light system, which emit visible light and/or invisible lightfor observation of the biological sample in the biological test.

Another objective of the present invention is to provide a multipleexciting light system, which has a plurality of light sources indifferent locations to enhance the excitation of fluorochrome inbiological sample.

According to the objectives of the present invention, the presentinvention provides a multiple exciting light system for a test of abiological sample labeled with fluorochrome, including a case, in whicha sample table, a first light source module, a second light sourcemodule, and filter unit are provided. The case has a chamber. The sampletable is provided in the chamber of the case to put the biologicalsample thereon. The first light source module is provided in the chamberof the case to emit a first light with a first wavelength, wherein thefirst light is visible light; and the second light source module isprovided in the chamber of the case to emit a second light with a secondwavelength, wherein the first light is visible light or invisible light.The first light from the first light source module and the second lightfrom the second light source module excite the fluorochrome in thebiological sample at the same time to generate a third light with athird wavelength by fluorescence resonance superposition energytransfer, FRET. The filter unit is provided in the chamber of the caseabove the sample table to filter noise of the third light out to form aclear third light.

In comparison with the prior art, the present invention provides themultiple exciting light system to emit visible light and/or invisiblelight. The visible and invisible light has multiple wavelengths whichexcites the fluorochrome -labeled biological sample to emit strongfluorescence. In an embodiment, the present invention further includes afilter unit to filter the light. In an embodiment, the present inventioncatches images before and after test to adjust brightness, whitebalance, or contrast of the biological sample's image.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sectional view of the multiple exciting light system of afirst preferred embodiment of the present invention;

FIG. 2 is a sketch diagram of the first preferred embodiment of thepresent invention, showing the arrangement of the first light source andthe second light source;

FIG. 3 is a sectional view of the multiple exciting light system of asecond preferred embodiment of the present invention;

FIG. 4 is a sketch diagram, showing the excitation of fluorochrome;

FIG. 5 is a sketch diagram, showing the generation of the third light;

FIG. 6 is a sectional view of the multiple exciting light system of athird preferred embodiment of the present invention;

FIG. 7 is a sectional view of the multiple exciting light system of afourth preferred embodiment of the present invention;

FIG. 8 is a sectional view of the multiple exciting light system of afifth preferred embodiment of the present invention;

FIG. 9 is a sectional view of the multiple exciting light system of asixth preferred embodiment of the present invention;

FIG. 10 is a sectional view of the multiple exciting light system of aseventh preferred embodiment of the present invention; and

FIG. 11 to FIG. 14 shows the results of biological tests in differentsamples exposed under different light sources.

DETAILED DESCRIPTION OF THE INVENTION

As shown in FIG. 1, a multiple light system 100 of the first preferredembodiment of the present invention is to excite fluorochrome 2 (socalled fluorophore) added in a biological sample 2. The biologicalsample 2 may be gel electrophoresis of deoxyribonucleic acid (DNA),protein, or bio-materials.

The multiple exciting light system 100 includes a case 12, a sampletable 14, a first light source module 16, and a second light sourcemodule 18. The case 12 forms a chamber 122 therein. The sample table 14is provided in the chamber 122 of the case 12 to put the biologicalsample 4 thereon. The sample table 14 may be transparent or matted.

The first light source module 16 and the second light source module 18are provided in the chamber 122 under the sample table 14 to emit thefirst light FW and the second light SW to the biological sample 4through the sample table 14 respectively or in the same time. FIG. 2shows an arrangement of the first light source module 16 and the secondlight source module 18 of an embodiment of the present invention.

The first light FW is visible light with a first wavelength. In otherwords, the first wavelength is in a range between 380 nm (purple) and750 nm (red). In an embodiment, the first light FW is blue light, andthe first wavelength is between 435 nm and 480 nm. The first lightsource module 16 includes a plurality of light sources 162, and thelight sources 162 may be blue LEDs (light emitting diode) in the presentinvention.

The second light SW may be visible light or invisible light, and thesecond wavelength may be in a range of visible light's wavelength (380nm and 750 nm) and invisible light's wavelength (280 nm (farultraviolet) and 380 nm (near ultraviolet)), or it may be greater than750 nm (infrared). For example, the second light source module 18 may beUV lamp, green light tube, or black light tube. In an embodiment, thesecond light SW is ultraviolet, and the second wavelength is between 250nm and 400 nm, or the second light SW is green light, and the secondwavelength is between 577 nm and 492 nm. The first and the second lightsFW and SW excite the fluorochrome 2 in the same time, which generate aspecified third light with a third wavelength by fluorescence resonancesuperposition energy transfer.

When the first light source module 16 and the second light source module18 emit the first light FW and the second light SW to the fluorochrome 2in the same time, the fluorochrome 2 absorbs a first energy Eg1 of thefirst light FW and a second energy Eg2 of the second light SW. Eg1 andEg2 may be obtained from the equation: Eg=hv, wherein h is Planckconstant (6.626×10⁻³⁴J·s) and v is frequency. After that, thefluorochrome 2 will be excited and generate the third light with a thirdenergy Eg3 by fluorescence resonance energy transfer (FRET).

FIG. 3 shows a multiple exciting light system 10 of the second preferredembodiment of the present invention. In addition to the case 12, thesample table 14, the first light source module 16, and the second lightsource module 18, it further contains a filter unit 20. The filter unit20 is provided in the chamber 122 of the case 12 above the sample table14 to filter noise of the third light TW out to form a clear third lightTW′. For example, the filter unit 20 may be a filter film in ambercolor. In other words, the wavelength of the clear third light TW′ isstill within the wavelength range of the third light TW, and is closedto a single wavelength.

As shown in FIG. 4, when the fluorochrome 2 absorbs the first energy Eg1and the second energy Eg2, photons of the fluorochrome 2 will be excitedto an excitation state S1 from an emission state S0, as shown in FIG. 4(a). After several nano-seconds, the photons will fall to anotherexcitation state S1′, which is slightly lower than the excitation stateS1, and then fall back to the emission state S0 again so that thefluorochrome 2 emits the third light TW with the third energy Eg3.Because of energy loss and reduction of photon's energy, radiationwavelength is longer than excitation wavelength, and the differencetherebetween calls Stokes shift. In other words, the wavelength ofincident light is different from the wavelength of the fluorescencereleased from the fluorochrome 2. Besides, each fluorochrome 2 has aspecified characteristic wavelength so that we may find a fluorescenceemission maximum at a specified emission wavelength (the characteristicwavelength) in the emission spectrum of the fluorochrome 2, as shown inFIG. 4( b).

As shown in FIG. 5, the visible first light FW has a firstcharacteristic wavelength FW′ to excite the fluorochrome 2 to emit thethird light TW. For example, when we choose SYPRO RUBY to be thefluorochrome 2, the first light FW is blue light and the firstwavelength is between 435 nm and 480 nm, and the first characteristicwavelength FW′ is about 470 nm. As a result, the fluorochrome 2 emitsthe third light TW of 610 nm after it absorbs the first characteristicwavelength FW′.

The invisible second light SW further has a second characteristicwavelength SW′ to excite the fluorochrome 2 to emit the third light TW.When we choose SYPRO Ruby to be the fluorochrome 2, the second light SWis UV light and the second wavelength is between 250 nm to 400 nm, andthe second characteristic wavelength SW′ is about 290 nm. As a result,the fluorochrome 2 emits the third light TW of 610 nm after it absorbsthe second characteristic wavelength SW′.

Both of the visible light (the first light FW) and the invisible light(the second light SW) may excite the fluorochrome 2 to emit the thirdlight TW with the same wavelength (610 nm for example) in the same time.The third energy Eg3 of the third light TW is about equal to the sum ofthe first energy Eg1 and the second Eg2 of the first and the secondcharacteristic wavelengths FW′ and SW′ by superposition and transfer ofenergy. In other words, when the fluorochrome 2 absorbs two kinds ofenergy, the intensity of fluorescence released from the fluorochrome 2is much greater than that the fluorochrome 2 only absorbs single energy.

FIG. 6 shows a multiple exciting light system 101 of the third preferredembodiment of the present invention, including the case 12, the sampletable 14, the first light source module 16, the second light sourcemodule 18, and the filter unit 20, the same as above. In the presentembodiment, the first light source module 16 is under the sample table14, and the second light source module 18 is above the sample table 14.The second light source module 18 directly emits the second light SW tothe biological sample 4 on the sample table 14 in a direction divergingfrom a normal of the sample table 14. In the fourth preferred embodimentof the present invention, a multiple exciting light system 102 furtherincludes a diffusion unit 22 above the first light source module 16 todiffuse the first light FW from the first light source module 16 andform a surface first light FW′ as shown in FIG. 7.

FIG. 8 shows a multiple exciting light system 103 of the fifth preferredembodiment of the present invention, in which the second light sourcemodule 18 is under the sample table 14 and the first light source module16 is above the sample table 14. The first light source module 16 emitsthe first light FW directly to the biological sample 4 on the sampletable 14 in a direction diverging from a normal of the sample table 14.

As shown in FIG. 9, a multiple exciting light system 104 of the sixthpreferred embodiment of the present invention provides both the firstand the second light source modules 16, 18 above the sample table 14 toemit the first light FW and the second light SW directly to thebiological sample 4 on the sample table 14 in directions diverging froma normal of the sample table 14.

FIG. 10 shows a multiple exciting light system 105 of the sixthpreferred embodiment of the present invention. Besides the case 12, thesample table 14, the first light source module 16, the second lightsource module 18, and the filter unit 20, it further includes an imagecapture unit 24 above the filter unit 20 to catch images through thefilter unit 20. The image capture unit 24 may catch a background imageBIMG which is taken when the biological sample 4 is not put on thesample table 14 yet, and a biological sample image BSIMG which is takenwhen the biological sample 2 has been put on the sample table 14. Themultiple exciting light system 105 further includes a comparison unit 26connected to the image capture unit 24 to compare the biological sampleimage BSIMG with the background image BIMG and to form a detection imageDIMG according to the comparison result. The detection image DIMG showsthe difference between the biological sample image BSIMG and thebackground image BIMG. The detection image DIMG may show the differenceof brightness, white balance, or contrast.

Hereafter, we provide several test results to show the performance ofthe multiple exciting light system of the present invention in thebiological test. In the following tests, gel electrophoresis of DNA andsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) isselected.

In the first control group, serially diluted protein molecular weightmarkers are loaded on SDS-PAGE. In the first lane, ten microliters ofprotein molecular weight markers are loaded. In the second lane, fivemicroliters of protein molecular weight markers are loaded. In the thirdlane, two point five microliters of protein molecular weight markers areloaded. The following lane works in the same way as the previous.Besides, SDS-PAGE is stained by SYPRO Ruby. FIG. 11( a) shows theSDS-PAGE of the first control group exposed to single UV light; FIG. 11(b) shows the SDS-PAGE of the first control group exposed to single bluelight; and FIG. 11( c) shows the SDS-PAGE of the first control groupexposed to blue light and UV light. The result shows that SDS-PAGE inFIG. 11( c) is clearer for observation than FIG. 11( a) and FIG. 11( b).

In the second control group, serially diluted DNA are subjected toelectrophoresis on agarose gel lanes. In the first lane, five hundrednanograms of DNA are loaded. In the second lane, two hundred and fiftynanograms of DNA are loaded. In the third lane, two hundred and twentyfive nanograms of DNA are loaded. The following lane works in the sameway as the previous. Besides, DNA gel is stained by SYBER Green I. FIG.12( a) shows the DNA gel of the second control group exposed to singleUV light; FIG. 12( b) shows the DNA gel of the second control groupexposed to single blue light; and FIG. 12( c) shows the DNA gel of thesecond control group exposed to blue light and UV light. The resultshows that DNA gel in FIG. 12( c) is clearer for observation than FIG.12( a) and FIG. 12( b).

In the third control group, one hundred micrograms of bovine serumalbumin (BSA) is stained with SYPRO Ruby in a small and transparenttube. FIG. 13( a) shows the BSA of the third control group exposed tosingle UV light; FIG. 13( b) shows the BSA of the third control groupexposed to single blue light; and FIG. 13( c) shows the BSA of the thirdcontrol group exposed to blue light and UV light. The result shows thatBSA in FIG. 13( c) is clearer for observation than FIG. 13( a) and FIG.13( b).

In the fourth control group, ten micrograms of DNA is stained with SYBERGreen I in a small and transparent tube. FIG. 14( a) shows the DNA ofthe fourth control group exposed to single UV light; FIG. 14( b) showsthe DNA of the fourth control group exposed to single blue light; andFIG. 14( c) shows the DNA of the fourth control group exposed to bluelight and UV light. The result shows that DNA in FIG. 14( c) is clearerfor observation than FIG. 14( a) and FIG. 14( b). The present inventionprovides the multiple exciting light system to emit visible light andinvisible light in the biological test, so that fluorochrome in thebiological sample is excited for fluorescence resonance energy transferto generate a specified fluorescence. The present invention furtherprovides the filter unit to filter the noise out and enhance the signalof the biological sample. The present invention further catches theimage before and after test to adjust the brightness, white balance, orcontrast of the image of biological sample in accordance with thedifference between the images.

The description above is a few preferred embodiments of the presentinvention and the equivalence of the present invention is still in thescope of claim construction of the present invention.

What is claimed is:
 1. A multiple exciting light system for a test of abiological sample labeled with fluorochrome, comprising: a case having achamber; a sample table provided in the chamber of the case to put thebiological sample thereon; a first light source module provided in thechamber of the case to emit a first light with a first wavelength,wherein the first light is visible light; and a second light sourcemodule provided in the chamber of the case to emit a second light with asecond wavelength, wherein the first light is visible light or invisiblelight; wherein the first light from the first light source module andthe second light from the second light source module excite thefluorochrome in the biological sample at the same time to generate athird light with a third wavelength.
 2. The multiple exciting lightsystem as defined in claim 1, further comprising a filter unit in thechamber of the case, wherein the third light emits to the filter unit tobe filtered.
 3. The multiple exciting light system as defined in claim1, wherein the first light source module has a plurality of lightsources.
 4. The multiple exciting light system as defined in claim 3,wherein the light sources of the first light source module are lightemitting diodes.
 5. The multiple exciting light system as defined inclaim 4, further comprising a diffusion unit in the chamber of the caseto diffuse the first light from the first light source module.
 6. Themultiple exciting light system as defined in claim 3, wherein the firstlight has a first characteristic wavelength to excite the fluorochrometo generate the third light.
 7. The multiple exciting light system asdefined in claim 6, wherein the first wavelength of the first light isin a range between 435 nm and 480 nm.
 8. The multiple exciting lightsystem as defined in claim 7, wherein the fluorochrome is excited by thefirst light to generate the third light with the third wavelength of 610nm while the first characteristic wavelength of the first light is 470nm.
 9. The multiple exciting light system as defined in claim 1, whereinthe second light has a second characteristic wavelength to excite thefluorochrome to generate the third light.
 10. The multiple excitinglight system as defined in claim 9, wherein the second wavelength of thesecond light is in a range between 250 nm and 400 nm.
 11. The multipleexciting light system as defined in claim 10, wherein the fluorochromeis excited by the second light to generate the third light with thethird wavelength of 610 nm while the first characteristic wavelength ofthe first light is 290 nm.
 12. The multiple exciting light system asdefined in claim 11, wherein the second light source module has UV lamp,green light tube, or black light tube.
 13. The multiple exciting lightsystem as defined in claim 12, wherein the sample table is transparentor matted.
 14. The multiple exciting light system as defined in claim13, wherein the first light source module and the second light sourcemodule are under the sample table to emit the first light and the secondlight to the biological sample through the sample table.
 15. Themultiple exciting light system as defined in claim 13, wherein one ofthe first light source and the second light source is under the sampletable, and the other one is above the sample table.
 16. The multipleexciting light system as defined in claim 15, wherein the one of thefirst light source and the second light source above the sample tabledirectly emits the light to the biological sample in a directiondiverging from a normal of the sample table.
 17. The multiple excitinglight system as defined in claim 13, wherein both of the first lightsource and the second light source are above the sample table todirectly emit the first light and the second light to the biologicalsample in directions diverging from a normal of the sample table. 18.The multiple exciting light system as defined in claim 2, furthercomprising an image capture unit above the filter unit to catch imagesthrough the filter unit, wherein the images include a background imagewhich is taken when the biological sample is not put on the sample tableyet, and a biological sample image which is taken when the biologicalsample is put on the sample table.
 19. The multiple exciting lightsystem as defined in claim 18, further comprising a comparison unitconnected to the image capture unit to compare the biological sampleimage with the background image to form a detection image.
 20. Themultiple exciting light system as defined in claim 19, wherein thedetection image shows a difference of brightness, white balance, orcontrast.
 21. The multiple exciting light system as defined in claim 2,wherein the filter unit is an amber filter.
 22. The multiple excitinglight system as defined in claim 1, wherein the biological sample is gelelectrophoresis.